Melioidosis is a tropical infection caused by inoculation, inhalation, or ingestion of the Gram-negative soil saprophyte and Tier 1 select agent Burkholderia pseudomallei (Bps). Pneumonia is a common manifestation of infection, is frequently associated with septic shock, and confers a two-fold increase in mortality. Bps is intrinsically resistant to many common antibiotics. The standard therapies to which Bps is susceptible are ceftazidime and carbapenems yet patients may take days to defervesce once on treatment and need extended courses of therapy. Treatments for melioidosis have not changed substantially in decades and the case fatality rate in northeast Thailand has remained at 40% since 1989. New therapies are urgently needed; yet to develop effective therapies it is essential to advance understanding of the pathogen's ability to persist, especially in the lung. The multiple PIs of this application offer expertise in pulmonary host defenses (West) and in Burkholderia virulence factors (Schwarz). Together, these investigators have studied both Bps and the closely related bacterium Burkholderia thailandensis (Bt), a surrogate organism to Bps that is avirulent in humans and exempt from select agent restrictions. Bt/Bps, facultative intracellular bacteria, enter a wide variety of cell types and can escape the endosome into the cytosol. The type III secretion system (T3SS) is involved in cellular invasion and endosomal escape. Bt/Bps can induce cell-cell fusions in a type VI secretion system (T6SS)-mediated fashion that may facilitate intercellular spread. Bps can enter primary alveolar macrophages and bronchial epithelial cells in vitro, yet replication in alveolar macrophages is contained whereas replication in bronchial epithelial cells is unrestricted. However, the extent of intracellular parasitism by Bt/Bps, the cells that Bt/Bps infects in the lung, and the effect of T3SS and T6SS on these host-pathogen interactions have not been studied in vivo. The central hypothesis of this project is that Bps differentially parasitizes, replicates, and spreads intercellularly in specific pulmonary cell types in vivo; these processes are variably dependent on T3SS or T6SS, and they impede successful treatment. It is critical to test this hypothesis in order to develop new cell- or pathogen-targeted therapies for this infection. This will be accomplished using a novel dual fluorescent Bt reporter construct that signals when the bacterium is located within the cytoplasm of the host cell. The aims of the project are: 1) Determine whether specific cell types are differentially parasitized by Bt in the lung in vivo; 2) Determine whether T3SS and T6SS alter intracellular localization and the specific cells parasitized by Bt in the lung in vivo; 3) Determine whether standard treatment for melioidosis successfully kills intracellular Bt in the lung in vivo. The scientific value of this project is that it will advance understanding of the specific host cells parasitized by Bps in the lung in vivo and the role of T3SS and T6SS virulence factors on this interaction, evaluate how this intracellular lifestyle impacts current antibiotic treatment efficacy, and identify cellular or bacterial targets for future novel therapeutic development.